Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach. H. pylori infection is associated with an increased risk of cancer of the distal stomach and peptic ulcer disease. In previous studies, we have conducted detailed analyses of a secreted H. pylori toxin (VacA). The H. pylori genome contains three genes that are distantly related to vacA, and each encodes a protein >250 kDa in size. Similar to VacA, these VacA-like proteins are predicted to be secreted by a type V (autotransporter pathway). Bacterial proteins that are secreted by autotransporter pathways typically have important roles in the pathogenesis of infectious diseases. In contrast to VacA, which has been studied in great detail, thus far there has been very little study of H. pylori VacA-like proteins. Recent studies provide evidence that one of the VacA-like proteins (FaaA) localizes to flagella and has a role in H. pylori motility, and another VacA-like protein (ImaA) has anti-inflammatory activity. Hypotheses: The overarching hypotheses of this proposal are as follows: (i) the transcription of VacA-like proteins is tightly regulated in response to multiple stimuli, thereby allowing orchestrated expression of these in a manner that optimizes H. pylori colonization of the human stomach, and (ii) each of the VacA-like proteins has a specialized function designed to enhance H. pylori colonization of the stomach. Study Objectives: The long-term goals of this work are to understand the molecular mechanisms that allow H. pylori to colonize and persist in the human gastric mucosa, to understand the molecular mechanisms by which H. pylori infection leads to the development of gastric cancer or peptic ulcer disease, and to develop effective strategies for the prevention of gastric cancer and peptic ulcer disease. The specific objectives are (i) to define the mechanisms by which faaA and imaA are regulated; (ii) to define the roles of FaaA and ImaA in H. pylori colonization of the stomach and H. pylori-induced gastric disease; and (iii) to define mechanisms by which ImaA modulates H. pylori-induced inflammatory responses. Methods: To investigate the regulation of faaA and imaA expression, we will undertake in-depth studies of the promoter regions of these genes and identify regulatory systems that are responsible for the observed regulation of gene expression. We will investigate the roles of FaaA and ImaA in vivo through use of a system that allows expression of these proteins under control of an inducible promoter. Finally, we will use multiple approaches to investigate how the presence or absence of ImaA influences multiple phenotypes, including assembly of the cag type IV secretion system.